The phosphorylation state and tyrosine kinase activity of insulin receptors in subfractions of insulin-treated rat adipose cells have been studied. The results suggest that insulin receptors retain their kinase activity on internalization, indicating that the receptor kinase may possibly mediate insulin's effect while inside the cell. However, if the internalized receptor kinase mediates insulin's effect on glucose transport, only a portion of its maximum activity appears to be necessary for full transport stimulation (<20%). Further, the difference in kinase activity among subfractions suggests that the receptor kinase in the low-density microsomes may be in the process of deactivation. The effects of isoproterenol and insulin on the subcellular distribution and phosphorylation state of insulin receptors have been investigated in rat adipose cells. The results suggest that isoproterenol augments insulin's effect on receptor internalization, but reverses its stimulatory effect on receptor phosphorylation state and tyrosine kinase activity in plasma membranes; the latter effects may account for the decreased sensitivity of the glucose transport response to insulin.